Joe Dunne was left beaming as Colchester completed a smash-and-grab raid at Gillingham to steal all three points just two minutes from time through Andrew Bond.
The substitute scored the only goal to settle the contest after collecting a pass from fellow replacement Drey Wright as the Gills suffered a losing start on their return to League One.
U's boss Dunne, who started his playing career at Gillingham, said: "It's a difficult place to come and we knew that it would be a hard afternoon.
"Gillingham is a good club with good fans but I thought we did a job and we were tremendous - everyone played to their limit.
"We had to defend a lot from throws and set plays into the box but I thought that we deserved our win and I'm very pleased with how we went about our business.
"We analysed every goal they scored last year and long throws and corners were defended well against them, in a different division.
"We used our substitutes wisely and we had a plan and stuck to it.
"We had to be resilient at times and you have to earn the right to play in any game that you play.
"We had to scrap at times but the goal looked like it was coming and we managed to get in behind their back four.
"It's a good win for us but it's only the first game and we'll be back in on Sunday morning, getting ready for the Peterborough United cup tie on Tuesday night.
"We're focusing on one game at a time and we'll treat every game like a cup final this season and see where that takes us."
Gillingham striker Danny Kedwell went close in the opening stages with a first-time snap-shot in an even first half.
Colchester forward Jabo Ibehre fired over from five yards from Freddie Sears' cross before the Gills went even closer when Kedwell's spectacular long-range effort was pushed onto the crossbar by U's keeper Sam Walker.
Charlie Lee went close again for Gillingham when Walker repelled him at the near post and Cody McDonald was denied by Brian Wilson's last-ditch tackle in the area in the opening minute of the second half.
Nelson pushed away Marcus Bean's first-time shot on the hour but Gillingham looked more likely to break the deadlock as the clock ticked down.
Joe Martin's long-range attempt flashed inches wide before Bond slotted home from Drey Wright's pass with two minutes to go to win it for Colchester.
Martin Allen was understandably disappointed to come away empty handed after Bond's late strike.
He said: "I'm not sure we deserved to lose. Our lads need a bit of help so they can believe in themselves and win games at this level.
"We lost a key player after four minutes. Matt Fish had a fantastic season last year - it's his knee and he'll have a scan on Monday.
"What a game for young Stephen Butcher to make his debut - I'm really proud of him."